Biology of mammalian fertilization: role of the zona pellucida.

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Authors

J Dean

Rapid expression of fibronectin in the rabbit heart after myocardial infarction with and without reperfusion.

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The expression of fibronectin in the repair process after myocardial infarction was studied using two protocols of coronary occlusion in the rabbit: a permanent occlusion or 3 h of occlusion followed by reperfusion (too late for salvage). We found a rapid and progressive increase in cardiac fibronectin expression in the infarcted region of the ventricle. Steady-state mRNA levels for fibronectin increased 13- and 16-fold, respectively, in the permanent and reperfused infarcts 1 d postinfarction. Immunological detection of the protein with a polyclonal antibody against plasma fibronectin showed significant increases of the protein fibronectin in the infarcted myocardium by day 3 in the reperfused group and by day 5 in the permanent coronary occlusion group. Ribonuclease protection assays established the induction of EIIIB containing fibronectin mRNA in both models by day 1 and use of a monoclonal antibody showed an increase in the EIIIA isoform 2 d postinfarction. Increases in steady-state mRNA levels for several collagen types were found in both groups, but these changes occurred after those noted for fibronectin. Thus fibronectin mRNA and protein expression increased rapidly postinfarction suggesting a functional role in the repair process.

Authors

A A Knowlton, C M Connelly, G M Romo, W Mamuya, C S Apstein, P Brecher

31P nuclear magnetic resonance measurements of muscle glucose-6-phosphate. Evidence for reduced insulin-dependent muscle glucose transport or phosphorylation activity in non-insulin-dependent diabetes mellitus.

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To assess the rate-limiting step in muscle glycogen synthesis in non-insulin-dependent diabetes mellitus (NIDDM), the concentration of glucose-6-phosphate (G6P) was measured by 31P nuclear magnetic resonance (NMR) during a hyperglycemic-hyperinsulinemic clamp. Six subjects with NIDDM and six age weight-matched controls were studied at similar steady-state plasma concentrations of insulin (approximately 450 pmol/liter) and glucose (11 mmol/liter). The concentration of G6P in the gastrocnemius muscle was measured by 31P NMR. Whole-body oxidative and nonoxidative glucose metabolism was determined by the insulin-glucose clamp technique in conjunction with indirect calorimetry. Nonoxidative glucose metabolism which under these conditions is a measure of muscle glycogen synthesis (1990. N. Engl. J. Med. 322:223-228), was 31 +/- 7 mumol/(kg body wt-min) in the normal subjects and 13 +/- 3 mumol/(kg body wt-min) in the NIDDM subjects (P less than 0.05). The concentration of G6P was higher (0.24 +/- 0.02 mmol/kg muscle) in the normal subjects than in the NIDDM subjects (0.17 +/- 0.02, P less than 0.01). Increasing insulin concentrations to insulin 8,500 pmol/liter in four NIDDM subjects restored the glucose uptake rate and G6P concentrations to normal levels. In conclusion, the lower concentration of G6P in the diabetic subjects despite a decreased rate of nonoxidative glucose metabolism is consistent with a defect in muscle glucose transport or phosphorylation reducing the rate of muscle glycogen synthesis.

Authors

D L Rothman, R G Shulman, G I Shulman

Isoform-specific regulation of platelet-derived growth factor activity and binding in osteoblast-enriched cultures from fetal rat bone.

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In osteoblast-enriched cultures from fetal rat bone, the A-chain homodimer of platelet-derived growth factor (PDGF-AA) is less potent than the PDGF isoforms containing B chain subunits (PDGF-AB and PDGF-BB), but normal osteoblasts appear to synthesize only PDGF-A subunit mRNA and polypeptide. However, other agents may regulate PDGF-AA activity in skeletal tissue. Pretreatment of osteoblast-enriched cultures with interleukin 1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) synergistically enhanced the mitogenic effect of PDGF-AA coincident with increased binding site occupancy, but neither factor augmented PDGF-BB activity or binding. Polyacrylamide gel analysis showed 125I-PDGF-AA binding complexes predominantly at greater than 200 kD and faint labeling at 185 kD. After IL-1 alpha or TNF-alpha pretreatment, PDGF-AA binding increased at both sites, but this effect was more striking at 185 kD, which co-migrated with 125I-PDGF-BB-labeled complexes. PDGF-AA binding sites were rapidly lost by comparison to those for PDGF-BB in cycloheximide-treated cultures, but they remained relatively enhanced by IL-1 alpha and TNF-alpha pretreatment. These studies indicate that IL-alpha and TNF-alpha increase PDGF-AA binding and activity for osteoblasts by mechanisms that are at least in part independent of new receptor synthesis, and suggest regulatory events that could control how PDGF binding sites specifically recognize different ligands.

Authors

M Centrella, T L McCarthy, W F Kusmik, E Canalis

Direct contact between sympathetic neurons and rat cardiac myocytes in vitro increases expression of functional calcium channels.

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To test the hypothesis that direct contact between sympathetic neurons and myocytes regulates expression and function of cardiac Ca channels, we prepared cultures of neonatal rat ventricular myocytes with and without sympathetic ganglia. Contractile properties of myocytes were assessed by an optical-video system. Contractility-pCa curves showed a 60% greater increase in contractility for innervated myocytes compared with control cells at 6.3 mM [Ca]0 (n = 8, P less than 0.05). Cells grown in medium conditioned by growth of ganglia and myocytes were indistinguishable physiologically from control cells. [Bay K 8644]-contractility curves revealed a 60 +/- 10% enhancement of the contractility response at 10(-6) M for innervated cells compared with control cells. The increased response to Bay K 8644 was not blocked by alpha- or beta-adrenergic antagonists. Moreover, increased efficacy of Bay K 8644 was maintained for at least 24 h after denervation produced by removal of ganglia from the culture. Dihydropyridine binding sites were assessed with the L channel-specific radioligand 3[H]PN200-110. PN200-110 binding sites were increased by innervation (51 +/- 5 to 108 +/- 20 fmol/mg protein, P less than 0.01), with no change in KD. Peak current-voltage curves were determined by whole-cell voltage clamp techniques for myocytes contacted by a neuron, control myocytes, and myocytes grown in conditioned medium. Current density of L-type Ca channels was significantly higher in innervated myocytes (10.5 +/- 0.4 pA/pF, n = 5) than in control myocytes (5.9 +/- 0.3 pA/pF, n = 8, P less than 0.01) or myocytes grown in conditioned medium (6.2 +/- 0.2 pA/pF, n = 10, P less than 0.01). Thus, physical contact between a sympathetic neuron and previously uninnervated neonatal rat ventricular myocytes increases expression of functional L-type calcium channels as judged by contractile responses to Ca0 and Bay K 8644, as well as by electrophysiological and radioligand binding properties.

Authors

S Ogawa, J V Barnett, L Sen, J B Galper, T W Smith, J D Marsh

Protein C inhibitor in human body fluids. Seminal plasma is rich in inhibitor antigen deriving from cells throughout the male reproductive system.

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An assay was developed for the measurement of human protein C inhibitor antigen (PCI) in blood plasma and other biological fluids. Both native PCI, modified inhibitor, and complexes of inhibitor with activated protein C or plasma kallikrein could be measured with the assay. Inhibitor antigen concentrations were found to be very high in seminal plasma (greater than 200 mg/liter), more than 40 times the concentration of PCI found in blood plasma. The inhibitor in seminal plasma was unable to form complexes with activated protein C. Gel filtration and immunoblotting findings indicated that the inhibitor in seminal plasma is present in a high molecular mass complex or cleaved to its modified form. As PCI antigen was absent from seminal plasma of patients with dysfunctional seminal vesicles, the seminal vesicle glands would appear to be the major source of seminal plasma PCI, a conclusion supported by immunohistochemical demonstration of the presence of PCI epitopes in the secretory epithelium of the seminal vesicles. Specific PCI immunoreactivity was also shown to be present in the testes, the epididymis glands, and the prostate, suggesting the inhibitor to have a complex or multiple function in the male reproductive system. Conclusive evidence of a local synthesis of PCI in the four male sex glands was provided by Northern blot analysis of RNA from these organs.

Authors

M Laurell, A Christensson, P A Abrahamsson, J Stenflo, H Lilja

Immunohistochemical detection of advanced glycosylation end products in diabetic tissues using monoclonal antibody to pyrraline.

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Pyrraline is one of the major Maillard compounds resulting from the reaction of glucose with amino compounds at slightly acidic pH. For in vivo studies, monoclonal pyrraline antibodies were raised after immunization of Balb/c mice with keyhole limpet hemocyamin-caproyl pyrraline conjugate. Of 660 hybridoma clones from one donor, 260 produced an antibody to the free hapten, two of which named Pyr-A and Pyr-B also cross-reacted with L-lysyl pyrraline. Using Pyr-B antibody and an ELISA, a gradual increase in pyrraline immunoreactivity was observed in serum albumin incubated with glucose or 3-deoxyglucosone. Plasma pyrraline levels increased fourfold (P less than 0.001) in Sprague-Dawley rats upon induction of diabetes with streptozotocin and were twofold increased in randomly selected plasmas from diabetic humans. Highly specific pyrraline immunoreactivity was detected in sclerosed glomeruli from diabetic and old normal kidneys as well as in renal arteries with arteriolosclerosis and in perivascular and peritubular sclerosed extracellular matrix and basement membranes. The preferential localization of pyrraline immunoreactivity in the extracellular matrix strengthens the notion that the advanced glycosylation reaction may contribute to decreased turnover and thickening of the extracellular matrix in diabetes and aging.

Authors

S Miyata, V Monnier

Intravascular filarial parasites inhibit platelet aggregation. Role of parasite-derived prostanoids.

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The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Platelets do not adhere to blood-borne microfilariae, and thrombo-occlusive phenomena are not observed in patients with circulating microfilariae. We studied the ability of microfilariae to inhibit human platelet aggregation in vitro. Brugia malayi microfilariae incubated with human platelets caused dose-dependent inhibition of agonist-induced platelet aggregation, thromboxane generation, and serotonin release. As few as one microfilaria per 10(4) platelets completely inhibited aggregation of platelets induced by thrombin, collagen, arachidonic acid, or ionophore A23187. Microfilariae also inhibited aggregation of platelets in platelet-rich plasma stimulated by ADP, compound U46619, or platelet-activating factor. The inhibition required intimate proximity but not direct contact between parasites and platelets, and was mediated by parasite-derived soluble factors of low (less than 1,000 Mr) molecular weight that were labile in aqueous media and caused an elevation of platelet cAMP. Prior treatment of microfilariae with pharmacologic inhibitors of cyclooxygenase decreased both parasite release of prostacyclin and PGE2 and microfilarial inhibition of platelet aggregation. These results indicate that microfilariae inhibit platelet aggregation, via mechanisms that may include the elaboration of anti-aggregatory eicosanoids.

Authors

L X Liu, P F Weller

Cyclic nucleotides attenuate thrombin-evoked alterations in parameters of platelet Na/H antiport. The role of cytosolic Ca.

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In this work, we explored the role of cyclic nucleotides in modulating parameters of the Na/H antiport in human platelets. Sodium nitroprusside and iloprost, as well as cyclic nucleotide analogues, were used to raise cellular levels of cAMP and cGMP. Cyclic nucleotides reversed the thrombin-evoked alkaline shift in cytosolic pH set point and the activity of the Na/H antiport, concurrently with attenuation of thrombin-induced rise in cytosolic free Ca. No effect of cyclic nucleotides was observed in platelets not treated with thrombin, or platelets subjected to phorbol 12-myristate 13-acetate. cAMP did not reverse ionomycin-induced changes in the parameters of the Na/H antiport. Collectively, these observations indicate that cyclic nucleotides modulate the Na/H antiporter in human platelets through their effect on thrombin-evoked changes in cytosolic free Ca. Presumably, this effect holds for other agonists which stimulate phospholipase C, raise cytosolic-free Ca, and activate the Na/H antiport through protein kinase C dependent and protein kinase C-independent mechanisms.

Authors

M Kimura, N Lasker, A Aviv

Decreased serum insulin-like growth factor-I associated with growth failure in newborn lambs with experimental cyanotic heart disease.

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To determine whether chronic hypoxemia results in alterations in endocrine function that may contribute to growth failure, we measured growth hormone (GH), somatomedins (insulin-like growth factors I and II, IGF-I and IGF-2), hepatic growth hormone receptors, and circulating IGF-binding proteins IGFBP-3 and IGFBP-2 in 12 newborn lambs with surgically created pulmonic stenosis and atrial septal defect, and in 10 controls. During chronic hypoxemia (oxygen saturation of 60-74% for 2 wk), weight gain was 60% of control (hypoxemic, 135 +/- 20 vs. control, 216 +/- 26 g/d, P less than 0.02). IGF-I was decreased by 43% (hypoxemic 253.6 +/- 29.3 SE vs. control 448.0 +/- 75.5 ng/ml, P = 0.01), whereas GH was unchanged (19.9 +/- 5.1 vs. 11.9 +/- 3.0 ng/ml, NS). The increase in IGF-1 was associated with a decrease in IGFBP-3 (hypoxemic, 5.09 +/- 1.25 vs. control, 11.2 +/- 1.08 arbitrary absorbency units per mm (Au.mm), P less than 0.01), and increase in IGFBP-2 (0.47 +/- 0.03 vs. 0.19 +/- 0.13 Au.mm, P less than 0.05), but no significant downregulation of hepatic GH receptors (hypoxemic, 106.1 +/- 20.1 vs. control, 147.3 +/- 25.9 fmol/mg, NS). Thus, chronic hypoxemia in the newborn is associated with a decrease in IGF-I and IGFBP-3 in the face of normal GH. This suggests peripheral GH unresponsiveness, similar to protein-calorie malnutrition or GH receptor deficiency dwarfism, but mediated at a level distal to the hepatic GH receptor.

Authors

D Bernstein, J R Jasper, R G Rosenfeld, R L Hintz

Regulatory effects of the saturated fatty acids 6:0 through 18:0 on hepatic low density lipoprotein receptor activity in the hamster.

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The plasma concentration of cholesterol carried in low density lipoproteins is principally determined by the level of LDL receptor activity (Jm) and the LDL-cholesterol production rate (Jt) found in animals or man. This study delineates which saturated fatty acids alter Jm and Jt and so increase the plasma LDL-cholesterol level. Jm and Jt were measured in vivo in hamsters fed a constant level of added dietary cholesterol (0.12%) and triacylglycerol (10%), where the triacylglycerol contained only a single saturated fatty acid varying in chain length from 6 to 18 carbon atoms. After feeding for 30 d, the 12:0, 14:0, 16:0, and 18:0 fatty acids, but not the 6:0, 8:0, and 10:0 compounds, became significantly enriched in the liver total lipid fraction of the respective groups fed these fatty acids. However, only the 12:0, 14:0, and 16:0 fatty acids, but not the 6:0, 8:0, 10:0, and 18:0 compounds, suppressed Jm, increased Jt, and essentially doubled plasma LDL-cholesterol concentrations. Neither the 16:0 nor 18:0 compound altered rates of cholesterol synthesis in the extrahepatic organs, and both lowered the hepatic total cholesterol pool. Thus, the different effects of the 16:0 and 18:0 fatty acids could not be attributed to a difference in cholesterol delivery to the liver. Since these changes in LDL kinetics took place without an apparent alteration in external sterol balance, the regulatory effects of the 12:0, 14:0, and 16:0 fatty acids presumably are mediated through some change in a putative intrahepatic regulatory pool of sterol in the liver.

Authors

L A Woollett, D K Spady, J M Dietschy

Beta-adrenergic upregulation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells.

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We used the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein to monitor the recovery of the intracellular pH (pHi) of rat parotid acini from an NH4(+)-induced alkaline load. This recovery was markedly inhibited by the loop diuretic bumetanide and by Cl- removal, indicating that it is largely due to NH4+ entry via the basolateral Na(+)-K(+)-2Cl- cotransporter. The rate of recovery of pHi was enhanced threefold by pretreatment (37.5 s) with isoproterenol (K1/2 = 21.5 nM) or norepinephrine (in the presence of phentolamine), and blocked by the beta 1-specific antagonist atenolol, indicating an upregulation of cotransport activity by beta 1-adrenergic stimulation. The effect of isoproterenol was prevented by protein kinase inhibitors and mimicked by cAMP analogues, and by maneuvers known to increase cytosolic cAMP levels in these cells, consistent with the involvement of protein kinase A. Physiologically, such an upregulation of the acinar Na(+)-K(+)-2Cl- cotransporter would lead to an increase in acinar chloride uptake across the basolateral membrane, and consequently, an increase in overall chloride and fluid secretion. Prevention of this upregulation by beta-blockers and possibly by other commonly used clinical agents may account for the dry mouth and dry eyes experienced by some patients taking these medications.

Authors

M Paulais, R J Turner

cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia.

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Adenosine 3',5'-cyclic monophosphate stimulates chloride (Cl-) secretion across airway epithelia. To determine whether cAMP also stimulates HCO3- secretion, we studied cultured canine and human airway epithelial cells bathed in a HCO3-/CO2-buffered, Cl(-)-free solution. Addition of forskolin stimulated an increase in short-circuit current that was likely a result of bicarbonate secretion because it was inhibited by a HCO3(-)-free solution, by addition of the carbonic anhydrase inhibitor, acetazolamide, or by mucosal addition of the anion channel blocker, diphenylamine 2-carboxylate. The current was dependent on Na+ because it was inhibited by removal of Na+ from the submucosal bathing solution, by addition of the Na+ pump inhibitor, ouabain, or by addition of amiloride (1 mM) to the submucosal solution. An increase in cytosolic Ca2+ produced by addition of a Ca2+ ionophore also stimulated short-circuit current. These data suggest that cAMP and Ca2+ stimulate HCO3- secretion across airway epithelium, and suggest that HCO3- leaves the cell across the apical membrane via conductive pathways. These results may explain previous observations that the short-circuit current across airway epithelia was not entirely accounted for by the sum of Na+ absorption and Cl- secretion. The cAMP-induced secretory response was absent in cystic fibrosis (CF) airway epithelial cells, although Ca(2+)-stimulated secretion was intact. This result suggests that HCO3- exist at the apical membrane is through the Cl- channel that is defectively regulated in CF epithelia. These results suggest the possibility that a defect in HCO3- secretion may contribute to the pathophysiology of CF pulmonary disease.

Authors

J J Smith, M J Welsh

Proliferation-dependent HIV-1 infection of monocytes occurs during differentiation into macrophages.

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Requirements for the establishment of productive infection with the human immunodeficiency virus type 1 (HIV-1) in primary monocytes were investigated. In vitro, monocytes rendered susceptible for infection after at least a 2-d culture, but when cultured in the presence of differentiation-inducing agent IL-4, accelerated susceptibility was seen. Complete resistance to HIV-1 infection was observed in monocytes that had been treated for 5 d with rIL-4, and comparable results were obtained with other differentiation inducers such as dexamethasone or 1,25(OH)2 vitamin D3 (1,25(OH)2vitD3). The inhibition of productive infection was not caused by downregulation of CD4 expression or HIV-1 transcription, nor by intracellular accumulation of virions. Since treatment with rIL-4, dexamethasone, or 1,25(OH)2vitD3 also resulted in complete inhibition of monocyte proliferation, we studied whether establishment of productive infection in monocytes is proliferation dependent. Irradiation or mitomycin-C treatment within 24 h after inoculation prevented productive HIV-1 infection of monocytes, suggesting a proliferation-dependent step early in the virus replication cycle. Polymerase chain reaction (PCR) analysis revealed the presence of only incomplete proviral DNA species in non-proliferating monocytes, indicating restriction of viral replication at the level of reverse transcription. Thus, in analogy with HIV-1 infection of CD4+ T cells, proliferation of monocytes during differentiation into macrophages is a prerequisite for productive infection with HIV.

Authors

H Schuitemaker, N A Kootstra, M H Koppelman, S M Bruisten, H G Huisman, M Tersmette, F Miedema

Islet-reactive T cells are a marker of preclinical insulin-dependent diabetes.

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The destruction of pancreatic islet beta cells in insulin-dependent diabetes mellitus (IDDM) is thought to be T cell mediated. To directly identify islet-reactive T cells in asymptomatic, "preclinical" IDDM individuals with islet cell antibodies (ICA), proliferation of peripheral blood mononuclear cells (PBMC) was measured in the presence of sonicated fetal pig proislets. Stimulation indices (mean +/- SD) for [3H]thymidine uptake by PBMC cultured with sonicated proislets were: preclinical IDDM subjects (n = 22) 6.10 +/- 6.50, recent-onset IDDM subjects (n = 29) 3.66 +/- 3.35, Graves' disease subjects (n = 6) 2.17 +/- 0.93, scleroderma subjects (n = 4) 1.65 +/- 0.19 and normal control subjects (n = 14) 1.63 +/- 0.62. 68% (15/22) of preclinical IDDM, 41% (12/29) of recent-onset IDDM and 17% (1/6) of Graves' disease subjects had T cell reactivity greater than the mean + 2 SD of controls. T cell reactivity to proislets was tissue specific, and greater in magnitude and frequency than to human insulin. The majority of preclinical subjects with ICA greater than 20 Juvenile Diabetes Foundation (JDF) units (12/15, 80%) or antibodies to a 64-kD islet autoantigen (11/15, 73%) had significant T cell reactivity to proislets. ICA greater than 40 JDF units, a strong prognostic marker for progression to clinical IDDM, was an absolute index of T cell reactivity. Overall, the frequency of T cell reactivity in preclinical subjects, 68% (15/22), was comparable to that of ICA greater than 20 JDF units or 64-kD antibodies. Greater T cell reactivity to proislets in preclinical subjects accords with the natural history of autoimmune beta cell destruction. The direct assay of islet-reactive T cells in peripheral blood may have prognostic significance for the development of clinical IDDM and should facilitate identification of the primary target autoantigen(s).

Authors

L C Harrison, S X Chu, H J DeAizpurua, M Graham, M C Honeyman, P G Colman

Hormonal regulation of serum Lp (a) levels. Opposite effects after estrogen treatment and orchidectomy in males with prostatic carcinoma.

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Serum concentrations of lipoprotein (a) [Lp (a)] were determined in two groups of elderly males suffering from prostatic carcinoma, who were randomized to treatment with estrogen (n = 15) or orchidectomy (n = 16). Estrogen was given as oral ethinylestradiol, 150 micrograms daily, combined with intramuscular polyestradiol phosphate, 80 mg/mo. The baseline levels were similar in both groups, but 6 mo after initiation of therapy, serum Lp (a) levels were decreased approximately 50% in the estrogen-treated group (P less than 0.001) in contrast to a 20% increase (P less than 0.01) in the orchidectomized group. Concomitantly, LDL cholesterol decreased by 30% and HDL cholesterol increased by almost 60% in the estrogen-treated patients. There was no relationship between the change in LDL cholesterol and Lp (a) reduction. In conclusion, Lp (a) levels in males were found to drastically decrease upon estrogen treatment and to increase after orchidectomy, suggesting that sex hormones, and particularly estrogens, exert a regulatory role on the serum Lp (a) level in man.

Authors

P Henriksson, B Angelin, L Berglund

Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

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A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

Authors

L J Thomas, M Urakaze, R DeGasperi, T Kamitani, E Sugiyama, H M Chang, C D Warren, E T Yeh

Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero.

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Hemopoietic stem cells from human fetal liver were transplanted in utero into preimmune fetal sheep (48-54 days of gestation). The fate of donor cells was followed using karyotype analysis, by immunofluorescence labeling with anti-CD antibodies, and by fluorescent in situ hybridization using human-specific DNA probes. Engraftment occurred in 13 of 33 recipients. Of five live born sheep that exhibited chimerism, all expressed human cells in the marrow, whereas three expressed them in blood as well. Engraftment was multilineage (erythroid, myeloid, and lymphoid) and human hemopoietic progenitors (multipotent colony-forming units, colony-forming units-granulocyte, macrophage, and erythroid burst-forming units) capable of forming colonies in vitro were detected in all five lambs for greater than 2 yr. These progenitors responded to human-specific growth factors both in vitro and in vivo. Thus the administration of recombinant human IL-3 and granulocyte macrophage-colony-stimulating factor to chimeric sheep resulted in a 2.1-3.4-fold increase in the relative expression of donor (human) cells. These results demonstrate that the permissive environment of the preimmune fetal sheep provides suitable conditions for the engraftment and long-term multilineage expression of human hemopoietic stem cells in a large animal model. In this model, donor human cells appear to retain certain phenotypic and functional characteristics that can be used to manipulate the size of donor cell pool.

Authors

E D Zanjani, M G Pallavicini, J L Ascensao, A W Flake, R G Langlois, M Reitsma, F R MacKintosh, D Stutes, M R Harrison, M Tavassoli

Preferential mRNA expression of prostromelysin relative to procollagenase and in situ localization in human articular cartilage.

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An imbalance between extracellular proteinases and their inhibitors is thought to underlie cartilage degradation. In cultures of adult cartilage, prostromelysin mRNA levels were much higher than those for procollagenase and this differential was increased in cultures stimulated with IL-1 beta. Analysis of mRNA prepared from freshly isolated chondrocytes showed abundant amounts of prostromelysin mRNA in normal adult cartilage but low levels in the neonate. Not all adult cartilage may possess such high levels of prostromelysin mRNA, as the message levels in the cartilage remaining on late-stage osteoarthritic joints were lower than those in normal adult cartilage. Relative to prostromelysin mRNA, little procollagenase and TIMP mRNA were found in the adult cartilage. In situ hybridization revealed that metalloproteinase mRNAs were localized in chondrocytes of the superficial zone in adult cartilage. However, upon IL-1 beta treatment, chondrocytes in all cartilage zones were observed to express prostromelysin mRNA. Relative to the neonate, the normal adult cartilage appears to have a high degradative potential, if one accepts that steady-state mRNA levels reflect prostromelysin production. As the adult cartilage is not apparently undergoing rapid turnover, it would appear that control of prostromelysin activation may be the major regulatory step in stromelysin-induced cartilage degradation.

Authors

Q Nguyen, J S Mort, P J Roughley

Sub-minimal inhibitory concentrations of cefmetazole enhance serum bactericidal activity in vitro by amplifying poly-C9 deposition.

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Serum-resistant organisms grown in sub-minimal inhibitory concentrations (subMICs) of antibiotics in vitro may be rendered sensitive to complement-mediated, serum bactericidal activity. We measured 125I-C3 and 125I-C9 deposition on genetically serum resistant Salmonella montevideo SH5770 (SH5770) that was rendered serum sensitive by growth in sub-MICs of cefmetazole (CMZ), a parenteral, second generation, cephamycin-group antibiotic. Three times as much C3 and over six times as much C9 bound to SH5770 grown in one-fourth the MIC of CMZ compared to broth-grown bacteria. SDS-PAGE analysis and autoradiography showed that neither the ratio of C3b:iC3b (approximately 1:2.5) nor the nature of the C3-bacterial bond was changed by growing the organisms in CMZ. Large amounts of complement membrane attack complexes containing poly-C9 were seen only on CMZ-grown SH5770 by SDS-PAGE and autoradiography. Poly-C9 was also detected only on CMZ-grown bacteria by indirect immunofluorescence and ELISA using a murine monoclonal antibody directed against a neoantigen of poly-C9. Bacterial hydrophobicity increased after growth in CMZ, and transmission electron micrographs of CMZ-grown SH5770 showed cell wall disruption and blebbing. These results indicate that growth in subMICs of CMZ increases bacterial hydrophobic domains available for interacting with the membrane attack complex, C5b-9, allowing formation and stable insertion of bactericidal complexes containing poly-C9.

Authors

J E Schweinle, M Nishiyasu

Association of polar amino acids at position 26 of the HLA-DQB1 first domain with the anticentromere autoantibody response in systemic sclerosis (scleroderma).

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Abstract

HLA class II alleles (detected by DNA typing) were determined in 116 Caucasians with systemic sclerosis positive and negative for anticentromere autoantibodies (ACA). Significantly increased frequencies of HLA-DR5(DRw11) (P = 0.009) and the Dw13(DRB1*0403, *0407) subtypes of DR4 (probability corrected, Pc = 0.005) were seen in ACA positive patients, and HLA-DR1 and DRw8 were also increased. These findings appeared to reflect linkage disequilibrium of DR5(DRw11) and many DR4(Dw13) haplotypes with HLA-DQw7 and DR1 with DQw5. In fact, the presence of a DQB1 allele having a polar glycine or tyrosine at position 26 of the DQB1 first domain versus a hydrophobic leucine accounted for 100% of ACA positive Caucasian systemic sclerosis patients compared to 69% of the ACA negative SS patients (P = 0.0008) and 71% of Caucasian controls (P = 0.0003) as well as all 7 ACA patients of non-Caucasian background. Furthermore, the genotype frequency of DQB1 alleles lacking leucine at position 26 was 73% in ACA positive SS patients, compared to 42% of ACA negative patients (P = 1.2 x 10(-5)) and 38% of controls (P = 5.8 x 10(-7)). These data, then, suggest that the second hypervariable region of the HLA-DQB1 chain may form the candidate epitope associated with the ACA response.

Authors

J D Reveille, D Owerbach, R Goldstein, R Moreda, R A Isern, F C Arnett

Constitutive and modulated expression of the human alpha 1 antitrypsin gene. Different transcriptional initiation sites used in three different cell types.

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Abstract

alpha 1-Antitrypsin (alpha 1 AT) is plasma glycoprotein that constitutes the principle inhibitor of neutrophil elastase in tissue fluids. It has been considered a prototype for liver-derived acute phase proteins in that its concentration in plasma increases three- to fourfold during the host response to inflammation/tissue injury. However, recent studies have shown that alpha 1 AT is expressed in several types of extrahepatic cells, including mononuclear phagocytes and enterocytes, and that there are distinct transcriptional units used in hepatocytes and at least one extra-hepatic cell type, blood monocytes. In this study, we have used a combination of ribonuclease protection assays, primer elongation analysis, and transcriptional run-on assays to further characterize mechanisms of basal and modulated alpha 1 AT gene expression in hepatocytes, enterocytes, and macrophages. The hepatoma cell line HepG2, intestinal epithelial cell line Caco2, and primary cultures of human peripheral blood monocytes were used as examples of the cell types. The results indicate that there are three macrophage-specific transcriptional initiation sites upstream from a single hepatocyte-specific transcriptional initiation site. Macrophages use these sites during basal and modulated expression. Hepatoma cells use the hepatocyte-specific transcriptional initiation site during basal and modulated expression but also switch on transcription from the upstream macrophage transcriptional initiation sites during modulation by the acute phase mediator interleukin 6 (IL-6). Caco2 cells use the hepatocyte-specific transcriptional initiation site during basal expression. There is a marked increase in the use of this site and an increase in the rate of transcriptional elongation of alpha 1 AT mRNA during differentiation of Caco2 cells from crypt-type to villous-type enterocytes. Caco2 cells also switch on transcription from the upstream macrophage transcriptional initiation sites during modulation by IL-6. These results provide further evidence that there are differences in the mechanisms of constitutive and regulated expression of the alpha 1 AT gene in at least three different cell types, HepG2-derived hepatocytes, Caco2-derived enterocytes and mononuclear phagocytes.

Authors

W Hafeez, G Ciliberto, D H Perlmutter

Human natural anti-Gal IgG regulates alternative complement pathway activation on bacterial surfaces.

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Abstract

One percent of circulating IgG in humans recognizes galactose alpha 1,3 galactose residues (anti-Gal) and is synthesized in response to stimulation by enteric bacteria. In this study, we found that the prevalence of binding of anti-Gal to blood isolates is significantly higher than its binding to normal stool isolates. When anti-Gal bound onto the lipopolysaccharide of a representative blood isolate, Serratia marcescens #21, it blocked its alternative complement pathway (ACP) lysis and made the organism serum resistant. In contrast, when anti-Gal bound to the capsular polysaccharide of a serum sensitive Serratia, #7, it increased ACP killing of this strain. The mechanism of blockade of ACP lysis by anti-Gal did not involve a decrease in the number of C3 molecules deposited onto Serratia #21 or an inhibition of the binding of C3b to its LPS, nor did it change the iC3b and C3d degradation products of bound C3b or prevent membrane attack complex formation on this organism. Our findings suggest that the effect of anti-Gal on immune lysis is dependent on the bacterial outer membrane structure to which it binds. We postulate that anti-Gal may play a role in the survival of selected Enterobacteriacae in Gram-negative sepsis by blocking ACP-mediated lysis of such bacteria by the nonimmune host, and that this effect depends on where anti-Gal finds its epitope on the bacterial outer membrane.

Authors

R M Hamadeh, G A Jarvis, U Galili, R E Mandrell, P Zhou, J M Griffiss

Autoantibodies to the ribosomal P proteins react with a plasma membrane-related target on human cells.

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Abstract

Autoantibodies to ribosomal P-proteins are present in 12-16% of patients with systemic lupus erythematosus and are associated with neuropsychiatric disease. As the ribosomal P proteins are located in the cytoplasm, the pathogenic effects of their cognate autoantibodies are unclear. In this study affinity-purified anti-P autoantibodies were used to explore the cell surface of several types of human and animal cells. Immunofluorescence as well as EM immunogold analysis demonstrated, on the surface of human hepatoma cells, the presence of an epitope that is antigenically related to the immunodominant carboxy terminus of P-proteins. The presence of this epitope was also demonstrated on the surface of human neuroblastoma cells and, to a lesser extent, on human fibroblasts. Furthermore, the Western blot technique revealed in purified human and animal plasma membranes a 38-kD protein that is closely related or identical with ribosomal P0 protein. The availability of reactive P peptide on the surface of cells makes possible the direct effect of autoantibodies on the function and viability of cells that express this antigenic target. This delineates one of the possible impacts of anti-P antibodies in disease expression.

Authors

E Koren, M W Reichlin, M Koscec, R D Fugate, M Reichlin

Suramin, an experimental chemotherapeutic drug, activates the receptor for epidermal growth factor and promotes growth of certain malignant cells.

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Abstract

Previous studies have shown that suramin is capable of disrupting autocrine growth involving coexpression of platelet-derived growth factor and its receptors in a fibroblast model for mesenchymal oncogenesis. Suramin is currently in use as an experimental drug for the treatment of patients with epithelial cell tumors. In the present study, we have investigated the efficacy of suramin in a carcinoma model system. Our findings demonstrate that suramin enhances cell surface signaling in A431 cells by activating an autocrine loop involving the receptor for epidermal growth factor (EGFR). The mechanism of suramin action was shown to be indirect, not affecting the ability of ligand to bind and activate the EGFR. Instead, suramin induced the release of membrane-bound transforming growth factor alpha, thereby increasing its potential to activate cell surface EGFRs. Since suramin potently blocks tyrosine phosphorylation induced by platelet-derived growth factor but can activate the growth pathway regulated by the EGFR, biological responses of tumor cells to suramin treatment may differ dramatically.

Authors

M Cardinali, O Sartor, K C Robbins

Nitric oxide and angiotensin II. Glomerular and tubular interaction in the rat.

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Abstract

Nitric oxide (NO) has been proposed to modulate the renal response to protein as well as basal renal hemodynamics. We investigated whether NO and angiotensin II (AII) interact to control glomerular hemodynamics and absolute proximal tubular reabsorption (APR) during glycine infusion and in unstimulated conditions. In control rats, glycine increased single nephron GFR and plasma flow with no change in APR. The NO synthase blocker, NG-monomethyl L-arginine (LNMMA), abolished the vasodilatory response to glycine, possibly through activation of tubuloglomerular feedback due to a decrease in APR produced by LNMMA + glycine. Pretreatment with an AII receptor antagonist, DuP 753, normalized the response to glycine at both glomerular and tubular levels. In unstimulated conditions, LNMMA produced glomerular arteriolar vasoconstriction, decreased the glomerular ultrafiltration coefficient, and reduced single nephron GFR. These changes were associated with a striking decrease in APR. DuP 753 prevented both glomerular and tubular changes induced by LNMMA. In conclusion, NO represents a physiological antagonist of AII at both the glomerulus and tubule in both the basal state and during glycine infusion; and inhibition of NO apparently enhances or uncovers the inhibitory effect of AII on proximal reabsorption.

Authors

L De Nicola, R C Blantz, F B Gabbai

Evidence of a role for mesothelial cell-derived interleukin 8 in the pathogenesis of asbestos-induced pleurisy in rabbits.

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Abstract

Although acute asbestos-induced pleurisy is characterized by an influx of neutrophils, the identity of the factors that attract these cells to the pleural space and the source of the factors are unknown. We found that instillation of crocidolite asbestos into the pleural space of rabbits led to the appearance in pleural liquid of chemotactic activity for neutrophils, and that this chemotactic activity was inhibited significantly by a neutralizing antibody to human interleukin 8 (IL-8). Cultured rabbit pleural mesothelial cells incubated with crocidolite asbestos also released chemotactic activity for neutrophils, which was inhibited significantly by the anti-IL-8 antibody. To determine whether rabbit pleural mesothelial cells synthesize IL-8, we generated a probe for rabbit IL-8 mRNA by amplifying cDNA prepared from stimulated pleural mesothelial cells using the polymerase chain reaction (PCR) and primers based on homologous sequences in human and sheep IL-8 cDNAs. Homology-based PCR yielded a single cDNA fragment with a nucleotide sequence 88% identical to that of a corresponding region of human IL-8 cDNA. With the radiolabeled PCR product as a probe, we demonstrated rapid induction of IL-8 mRNA expression in pleural mesothelial cells exposed to asbestos. As expected, tumor necrosis factor-alpha also led to the appearance of IL-8 in the rabbit pleural space and stimulated cultured pleural mesothelial cells to synthesize and release IL-8. We conclude that asbestos directly stimulates pleural mesothelial cells to synthesize IL-8 and that mesothelial cell-derived IL-8 may play an important role in mediating asbestos-induced pleural inflammation.

Authors

A M Boylan, C Rüegg, K J Kim, C A Hébert, J M Hoeffel, R Pytela, D Sheppard, I M Goldstein, V C Broaddus

Conserved mechanism of negative gene regulation by extracellular calcium. Parathyroid hormone gene versus atrial natriuretic polypeptide gene.

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Abstract

We found that a negative calcium-responsive element (nCaRE) originally reported in the human parathyroid hormone gene is conserved among several genes. The results of the present study show that expression of one of the genes, the rat atrial natriuretic polypeptide gene, was negatively regulated in the heart by extracellular calcium by using an in vivo infusion system. Moreover, transfection of the cultured cells revealed that this DNA element conferred negative regulation by extracellular calcium on the reporter gene. It is suggested that there is a gene family whose expression is negatively regulated by extracellular calcium through this conserved DNA motif, nCaRE.

Authors

T Okazaki, K Ando, T Igarashi, E Ogata, T Fujita

Allelic polymorphisms of human Fc gamma receptor IIA and Fc gamma receptor IIIB. Independent mechanisms for differences in human phagocyte function.

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Abstract

Two different allelic polymorphisms among the isoforms of human Fc gamma receptors have been defined: the low-responder (LR)-high-responder (HR) polymorphism of huFc gamma RIIA expressed on both PMN and monocytes and the NA1-NA2 polymorphism of the neutrophil Fc gamma RIII (huFc gamma RIIIB). To address the issues of whether the LR-HR polymorphism has a significant impact on Fc gamma R-mediated functions in human blood cells and whether any differences in LR-HR might be related to higher Fc gamma R-mediated phagocytosis in NA1 donors, we examined Fc gamma R-specific binding and internalization by donors homozygous for the two huFc gamma RIIA alleles. PMN from LR homozygotes showed consistently higher levels of internalization of erythrocytes opsonized with pooled human IgG (E-hIgG). The absence of an LR-HR phagocytic difference with erythrocytes opsonized with either anti-Fc gamma RIIA MAb IV.3 or rabbit IgG, as opposed to E-hIgG, suggested that the Fc piece of the opsonin might be important for this LR-HR difference. Accordingly, we studied HR and LR homozygotes with human IgG subclass-specific probes. Both PMN (independent of huFc gamma RIIIB phenotype) and monocytes from LR donors bound and internalized erythrocytes coated with human IgG2 (E-hIgG2) efficiently, whereas phagocytes from HR donors did so poorly. E-hIgG2 internalization was completely abrogated by blockade of the ligand binding site of huFc gamma RIIA with IV.3 Fab, indicating that huFc gamma RIIA is essential for the handling of hIgG2 and that the mechanism of the LR-HR phagocytic difference is at the level of ligand binding to huFc gamma RIIA. In contrast, the difference in internalization of E-hIgG between NA1 and NA2 homozygous donors was independent of the huFc gamma RIIA phenotype and did not manifest differences in ligand binding. Thus, the two known allelic polymorphisms of human Fc gamma R have distinct and independent mechanisms for altering receptor function, which may influence host defense and immune complex handling.

Authors

J E Salmon, J C Edberg, N L Brogle, R P Kimberly

Ultraviolet-irradiated monocytes efficiently inhibit the intracellular replication of Mycobacterium avium intracellulare.

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Abstract

The purpose of this study was to evaluate the effect of ultraviolet (UV) radiation on the antimicrobial activities of monocytes for the intracellular pathogen Mycobacterium avium intracellulare (MAI). UV radiation augmented monocyte antimicrobial activity for MAI in a dose-dependent fashion. UVB doses of greater than or equal to 25 J/m2 resulted in a 50-100-fold reduction in MAI growth 7 d after initiation of culture. The increased monocyte antibacterial effect could be blocked by a plate glass filter, indicating that wavelengths within the UVB were responsible for the effect. UV radiation did not stimulate monocyte phagocytosis, and enhanced inhibition of MAI growth was observed in populations of adherent mononuclear cells that were devoid of T cells. This suggested that UV radiation acted directly to augment intrinsic monocyte antimicrobial activities. The administration of 8-methoxypsoralen plus UVA radiation to monocytes also augmented their antimicrobial activities against MAI. UV radiation thus may serve as a unique agent by which to evaluate the mechanisms by which mononuclear phagocytes control the growth of MAI.

Authors

W S Mirando, H Shiratsuchi, K Tubesing, H Toba, J J Ellner, C A Elmets

Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse B cells.

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Abstract

Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels, depolarization, and Ca2+ influx in B cells. Mouse islets were used to investigate whether glucose can still regulate insulin release when it cannot control ATP-sensitive K+ channels. Opening of these channels by diazoxide (100-250 mumol/liter) blocked the effects of glucose on B cell membrane potential (intracellular microelectrodes), free cytosolic Ca2+ (fura-2 method), and insulin release, but it did not prevent those of high K (30 mmol/liter). K-induced insulin release in the presence of diazoxide was, however, dose dependently increased by glucose, which was already effective at concentrations (2-6 mmol/liter) that are subthreshold under normal conditions (low K and no diazoxide). This effect was not accompanied by detectable changes in B cell membrane potential. Measurements of 45Ca fluxes and cytosolic Ca2+ indicated that glucose slightly increased Ca2+ influx during the first minutes of depolarization by K, but not in the steady state when its effect on insulin release was the largest. In conclusion, there exists a mechanism by which glucose can control insulin release independently from changes in K(+)-ATP channel activity, in membrane potential, and in cytosolic Ca2+. This mechanism may serve to amplify the secretory response to the triggering signal (closure of K(+)-ATP channels--depolarization--Ca2+ influx) induced by glucose.

Authors

M Gembal, P Gilon, J C Henquin

Effect of acute changes in glomerular filtration rate on Na+/H+ exchange in rat renal cortex.

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Abstract

Studies were undertaken in Munich-Wistar rats to assess the influence of changes in filtered bicarbonate (FLHCO3), induced by changes in GFR, on Na+/H+ exchange activity in renal brush border membrane vesicles (BBMV). Whole-kidney and micropuncture measurements of GFR, FLHCO3, and whole-kidney and proximal tubule HCO3 reabsorption (APRHCO3) were coupled with BBMV measurements of H+ gradient-driven 22Na+ uptake in each animal studied. 22Na+ uptake was measured at three Na+ concentration gradients to allow calculation of Vmax and Km for Na+/H+ exchange. GFR was varied by studying animals under conditions of hydropenia, plasma repletion, and acute plasma expansion. The increase in GFR, FLHCO3, and APRHCO3 induced by plasma administration correlated directly with an increase in the Vmax for Na+/H+ exchange in BBMV. The Km for sodium was unaffected. In the plasma-expanded rats, the Vmax for Na+/H+ exchange was 22% greater than in the hydropenic rats (P less than 0.025) whereas APRHCO3 was 86% greater (P less than 0.001). These results indicate that increases in FLHCO3, induced by acute increases in GFR, stimulate Na+/H+ exchange activity in proximal tubular epithelium. This stimulation is a mechanism which can, in part, account for the delivery dependence of proximal bicarbonate reabsorption.

Authors

D A Maddox, S M Fortin, A Tartini, W D Barnes, F J Gennari

Factors affecting hydrogen production and consumption by human fecal flora. The critical roles of hydrogen tension and methanogenesis.

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Abstract

We studied the influence of hydrogen tension (PH2) and methanogenesis on H2 production and consumption by human fecal bacteria. Hydrogen consumption varied directly with PH2, and methanogenic feces consumed H2 far more rapidly than did nonmethanogenic feces. At low PH2, H2 production greatly exceeded consumption and there was negligible accumulation of the products of H2 catabolism, methane and sulfide. Thus, incubation at low PH2 allowed the first reported measurements of absolute as opposed to net H2 production. Feces incubated at high and intermediate PH2 had a net H2 production of only 1/900 and 1/64 of absolute production. Glucose fermentation by fecal bacteria yielded an absolute H2 production of 80 ml/g, a value far in excess of that excreted by volunteers ingesting lactulose. We conclude that most H2 produced by colonic bacteria is consumed and methanogenesis and fecal stirring (via its influence on fecal PH2) are critical determinants of H2 consumption and, hence, net H2 production. Study of fecal samples from four subjects with low breath H2 excretion after lactulose showed that absolute H2 production was normal, and the low H2 excretion apparently reflected increased consumption due to rapid methanogenesis (two subjects) and decreased luminal stirring (two subjects).

Authors

A Strocchi, M D Levitt

CD23 antigen regulation and signaling in chronic lymphocytic leukemia.

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Abstract

B lymphocytes from patients with chronic lymphocytic leukemia (B-CLLs), strongly express the CD23 antigen, a surface marker with significant prognostic importance in this disease. Because we previously reported that IL-4 shows a poor capacity for CD23 expression on B-CLLs, we first examined the possible mechanisms underlying CD23 overexpression on B-CLLs and found that mitogen-activated CLL T cells release soluble factors that are capable, in synergy with IL-4, of strongly inducing CD23. Using neutralizing Abs, we noticed that the T-cell-derived enhancing activity is entirely ascribed to the combined effects of IFN gamma (potent inhibitor of CD23 on normal B cells), TNF alpha (which has no effect on normal B cells), and IL-2 (which has a slight enhancing effect on both CLL and normal B cells). Furthermore, recombinant IFN gamma as well as IFN alpha, TNF alpha, and IL-2 (but not IL-3, IL-5, IL-6, IL-7, and lymphotoxin) significantly enhance CD23 protein and mRNA expression on B-CLLs, in the presence or absence of IL-4. Inasmuch as optimal CD23 expression absolutely requires the combination of IFN gamma, IL-2, TNF alpha (the production of which is increased in CLL disease), and IL-4, it was relevant to show that IL-4 mRNA is indeed expressed in fresh T-CLL cells. We next examined the possible role of CD23 in the regulation of B-CLL proliferation. Signaling through CD23 via ligation of the antigen by F(ab')2 anti-CD23 MAb but not Fab fragments inhibits the cytokine-induced B-CLL DNA synthesis. It is concluded that the CD23 gene is abnormally regulated in B-CLL disease and that cross-linking of CD23 molecule delivers a negative growth signal to the leukemic B cells.

Authors

S Fournier, G Delespesse, M Rubio, G Biron, M Sarfati

Interferon-gamma inhibits scavenger receptor expression and foam cell formation in human monocyte-derived macrophages.

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Abstract

The scavenger receptor (ScR) mediates uptake of chemically modified low density lipoprotein (LDL) by human monocyte-derived macrophages. It is not down-regulated by high intracellular cholesterol levels, and exposure of macrophages to acetylated or oxidized LDL therefore leads to foam cell development. The hypothesis that this represents an important mechanism for intracellular cholesterol accumulation in atherosclerosis is supported by the finding of ScR expression in foam cells of atherosclerotic plaques. T lymphocytes are also present in such plaques and it is known that T cell products regulate macrophage activation. We have therefore studied the effect of interferon-gamma (IFN gamma), a lymphokine secreted by activated T lymphocytes, on the expression of ScR in human monocyte-derived macrophages. Binding and uptake of acetylated LDL were significantly reduced in macrophages exposed to recombinant IFN gamma or IFN gamma-containing lymphocyte-conditioned media. Competition experiments showed that the IFN gamma-regulated binding and uptake of acetylated LDL was mediated via ScR. IFN gamma exerted its effect on the saturable binding of acetylated LDL by reducing the number of cell surface binding sites without significantly affecting the affinity between acetylated LDL and its receptor. Northern analysis revealed that the type I ScR mRNA was significantly reduced in IFN gamma-treated cells. Finally, IFN gamma treatment reduced intracellular cholesteryl ester accumulation and inhibited the development of foam cells in the cultures. In conclusion, our data show that IFN gamma blocks the development of macrophage-derived foam cells by inhibiting expression of ScR. This suggests that macrophage-T lymphocyte interactions may reduce intracellular cholesterol accumulation in the atherosclerotic plaque.

Authors

Y J Geng, G K Hansson

Immunoglobulin heavy chain gene expression in peripheral blood B lymphocytes.

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Abstract

cDNA libraries for IgM heavy chain variable regions were prepared from unmanipulated peripheral blood lymphocytes of two healthy people. Partial sequencing of 103 clones revealed VH gene family use and complete CDR3 and JH sequences. The libraries differed in the two subjects. In one person's cDNA the VH5 family was overexpressed and the VH3 family underexpressed relative to genomic complexity. In the second person's cDNA, VH3 was most frequently expressed. In both libraries, JH4 was most frequent. VH segments of several clones were closely related to those in fetal repertoires. However, there was also evidence of mutation in many cDNAs. Three clones differed from the single nonpolymorphic VH6 germline gene by 7-13 bases. Clones with several differences from VH5 germline gene VH251 were identified. CDR3 segments were highly diverse. JH portions of several CDR3's differed from germline JH sequences. 44% of the clones had DH genes related to the DLR and DXP families, most with differences from germline sequences. In 11 DLR2-related sequences, several base substitutions could not be accounted for by polymorphism. Thus, circulating IgM-producing B cell populations include selected clones, some of which are encoded by variable region gene segments that have mutated from the germline form.

Authors

C Huang, A K Stewart, R S Schwartz, B D Stollar

Predictive testing for multiple endocrine neoplasia type 1 using DNA polymorphisms.

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Abstract

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominantly inherited predisposition to neoplastic lesions of the parathyroids, pancreas, and the pituitary. We have previously located the predisposing genetic defect to the long arm of chromosome 11 by genetic linkage. In this study, 124 members of six MEN1 families, including 59 affected individuals, were genotyped for restriction fragment length polymorphisms with different DNA probes, and the genetic linkage between these marker systems and MEN1 was determined. 13 marker systems (17 DNA probes) were found to be linked to MEN1. These markers are located within a region on chromosome 11 spanning 14% meiotic recombinations, with the MEN1 locus in the middle. Four of the marker systems are on the centromeric side of MEN1, and four on the telomeric side, based on meiotic crossovers. The remaining five DNA probes are closely linked to MEN1, with no crossovers in our set of families. The 13 marker systems can be used for an accurate and reliable premorbid test for MEN1. In most clinical situations it is possible to identify a haplotype of this part of chromosome 11 with the mutant MEN1 allele in the middle. The calculated predictive accuracy is greater than 99.5% if three such marker systems are informative. Therefore, genetic linkage testing can be used for informed genetic counseling in MEN1 families, and to avoid unnecessary biochemical screening programs.

Authors

C Larsson, J Shepherd, Y Nakamura, C Blomberg, G Weber, B Werelius, N Hayward, B Teh, T Tokino, B Seizinger

Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation.

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Abstract

Platelet activation by thrombin is critical for hemostasis and thrombosis. Structure-function studies with a recently cloned platelet thrombin receptor suggest that a hirudin-like domain in the receptor's extracellular amino terminal extension is a thrombin-binding determinant important for receptor activation. We now report that a peptide antiserum to this domain is a potent and specific antagonist of thrombin-induced platelet activation. This study demonstrates that the cloned platelet thrombin receptor is necessary for platelet activation by thrombin, and provides a strategy for developing blocking monoclonal antibodies of potential therapeutic value.

Authors

D T Hung, T K Vu, V I Wheaton, K Ishii, S R Coughlin

Zidovudine induces molecular, biochemical, and ultrastructural changes in rat skeletal muscle mitochondria.

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Abstract

Zidovudine (AZT) inhibits HIV-1 replication in AIDS. A limiting side effect is AZT-induced toxic myopathy. Molecular changes in a rat model of AZT-induced toxic myopathy in vivo helped define pathogenetic molecular, biochemical, and ultrastructural toxic events in skeletal muscle and supported clinical and in vitro findings. After 35 d of AZT treatment, selective changes in rat striated muscle were localized ultrastructurally to mitochondria, and included swelling, cristae disruption, and myelin figures. Decreased muscle mitochondrial (mt) DNA, mtRNA, and decreased mitochondrial polypeptide synthesis in vitro were found in parallel. Mitochondrial molecular changes occurred in absence of altered abundance of cytosolic glyceraldehyde-3-phosphate dehydrogenase, or sarcomeric mitochondrial creatine kinase mRNAs. Quadriceps mitochondrial DNA polymerase gamma activity was similar in both AZT-treated and control rats. In vivo findings with rats support the hypothesis that AZT-induced inhibition of mtDNA replication has an effect of depressing the abundance of striated muscle mtDNA, mtRNA, and mitochondrial polypeptide synthesis. This experimental approach may be useful to examine mitochondrial or toxic myopathies.

Authors

W Lewis, B Gonzalez, A Chomyn, T Papoian

Reinfection of liver graft by hepatitis C virus after liver transplantation.

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Abstract

We have investigated hepatitis C virus (HCV) viremia before and after orthotopic liver transplantation (OLT). 38 patients were examined; 16 were anti-HCV positive and 22 anti-HCV negative pre-OLT in a RIBA-2 test (Ortho Diagnostic Systems Inc., Westwood, MA). HCV-RNA was detected using a modified nested polymerase chain reaction in 14/38 and 10/38 patients before and after OLT, respectively. 7 of these 14 subjects who were HCV-RNA positive before OLT were also positive for serum hepatitis B surface antigen. After OLT, six patients became HCV-RNA positive, likely as a result of transfusions, while four developed a probable recurrence of HCV infection. Infection of the liver graft by the same strain of HCV was indeed demonstrated by sequence analysis of a hypervariable domain (in the envelope region) in two cases. This establishes the possibility of HCV recurrence and shows the usefulness of polymerase chain reaction as the only assay currently capable of identifying HCV infection after OLT.

Authors

C Féray, D Samuel, V Thiers, M Gigou, F Pichon, A Bismuth, M Reynes, P Maisonneuve, H Bismuth, C Bréchot